Hsp90 Inhibitory Activity Research Articles

Triazole Containing Novobiocin and Biphenyl Amides as Hsp90 C-Terminal Inhibitors.

Hsp90 C-terminal inhibitors are advantageous for the development of new cancer chemotherapeutics due to their ability to segregate client protein degradation from induction of the prosurvival heat shock response, which is a major detriment associated with Hsp90 N-terminal inhibitors under clinical investigation. Based upon prior SAR trends, a 1,2,3-triazole side chain was placed in lieu of the aryl side chain and attached to both the coumarin and biphenyl scaffold. Antiproliferative studies against SKBr3 and MCF-7 breast cancer cell lines demonstrated these triazole-containing compounds to exhibit improved activity. These compounds were shown to manifest Hsp90 inhibitory activity through Western blot analysis and represent a new scaffold upon which more potent inhibitors can be pursued.

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.

Synthesis and Structure–Activity Relationships of EGCG Analogues, a Recently Identified Hsp90 Inhibitor

Epigallocatechin-3-gallate (EGCG), the principal polyphenol isolated from green tea, was recently shown to inhibit Hsp90; however, structure-activity relationships for this natural product have not yet been produced. Herein, we report the synthesis and biological evaluation of EGCG analogues to establish structure-activity relationships between EGCG and Hsp90. All four rings as well as the linker connecting the C- and the D-rings were systematically investigated, which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy over EGCG. Antiproliferative activity of all the analogues was determined against MCF-7 and SKBr3 cell lines and Hsp90 inhibitory activity of the four most potent analogues was further evaluated by Western blot analyses and degradation of Hsp90-dependent client proteins. The prenyl-substituted aryl ester of 3,5-dihydroxychroman-3-ol ring system was identified as a novel scaffold that exhibits Hsp90 inhibitory activity.

Structure-activity relationship (SAR) of withanolides to inhibit Hsp90 for its activity in pancreatic cancer cells

Withaferin A (WA), a naturally occurring steroidal lactone, directly binds to Hsp90 and leads to the degradation of Hsp90 client protein. The purpose of this study is to investigate the structure activity relationship (SAR) of withanolides for their inhibition of Hsp90 and anti-proliferative activities in pancreatic cancer cells. In pancreatic cancer Panc-1 cells, withaferin A (WA) and its four analogues withanolide E (WE), 4-hydroxywithanolide E (HWE), 3-aziridinylwithaferin A (AzWA) inhibited cell proliferation with IC50 ranged from 1.0 to 2.8 μM. WA, WE, HWE, and AzWA also induced caspase-3 activity by 21-, 6-, 11- and 15-fold, respectively, in Panc-1 cells, while withaperuvin (WP) did not show any activity. Our data showed that WA, WE, HWE, and AzWA, but not WP, all directly bound to Hsp90 and induced Hsp90 aggregation,hence inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins Akt and Cdk4 through proteasome-dependent pathway in pancreatic cancer cells. However, only WA, HWE and AzWA disrupted Hsp90-Cdc37 complexes but not WE and WP. SAR study suggested that the C-5(6)-epoxy functional group contributes considerably for withanolide to bind to Hsp90, inhibit Hsp90 chaperone activity, and result in Hsp90 client protein depletion. Meanwhile, the hydroxyl group at C-4 of ring A may enhance withanolide to inhibit Hsp90 activity and disrupt Hsp90-Cdc37 interaction. These SAR data provide possible mechanisms of anti-proliferative action of withanolides.

Anti-Inflammatory Activity of Hsp90 Inhibitors in the Human Vasculature

Anti-Inflammatory Activity of Hsp90 Inhibitors in the Human Vasculature

Biological Evaluation of 3-Acyl-2-Arylamino-1,4-Naphthoquinones as Inhibitors of Hsp90 Chaperoning Function

Hsp90 is a chaperone that plays a key function in cancer cells by stabilizing proteins responsible of cell growth and survival. Disruption of the Hsp90 chaperone machinery leads to the proteasomal degradation of its client proteins. Hsp90 appears then as an attractive target for the development of new anticancer molecules. We have shown that ascorbate- driven menadione-redox cycling inhibits Hsp90 activity by provoking an N-terminal cleavage of the protein, inducing the degradation of several of its client proteins. Since the mechanism involves an oxidative stress, we explored the effect of a series of diverse donor-acceptor 3-acyl-2-phenylamino 1,4-naphthoquinones on Hsp90 integrity, in the presence of ascorbate. Results show that quinone-derivatives that bear two electroactive groups (namely quinone and nitro) exhibit the highest inhibitory activity (Hsp90 cleavage and cell death). The biological activity of the series mainly relies on their redox capacity and their lipophilicity, which both modulate the ability of these compounds to induce a cytotoxic effect in K562 cells. As observed with other redox cycling quinones, the protein cleavage is blocked in the presence of N-terminal Hsp90 inhibitors suggesting that the availability or occupancy of nucleotide binding site in the N-terminal pocket of Hsp90 plays a critical role. In addition the survival of cancer cells and their metabolic and redox homeostasis were strongly impaired by the presence of ascorbate. Since these effects were similar to that obtained by ascorbate/menadione and they were blocked by the antioxidant N-acetylcyteine (NAC), it appears that oxidative stress is a major component of this cytotoxicity. Keywords: Cancer cell death, Hsp90, oxidative stress, protein cleavage, quinones, redox cycling, lipophilicity, cancer cells, N-acetylcyteine (NAC), cytotoxic, client proteins.

Biological Evaluation of 3-Acyl-2-Arylamino-1,4-Naphthoquinones as Inhibitors of Hsp90 Chaperoning Function

Hsp90 is a chaperone that plays a key function in cancer cells by stabilizing proteins responsible of cell growth and survival. Disruption of the Hsp90 chaperone machinery leads to the proteasomal degradation of its client proteins. Hsp90 appears then as an attractive target for the development of new anticancer molecules. We have shown that ascorbate- driven menadione-redox cycling inhibits Hsp90 activity by provoking an N-terminal cleavage of the protein, inducing the degradation of several of its client proteins. Since the mechanism involves an oxidative stress, we explored the effect of a series of diverse donor-acceptor 3-acyl-2-phenylamino 1,4-naphthoquinones on Hsp90 integrity, in the presence of ascorbate. Results show that quinone-derivatives that bear two electroactive groups (namely quinone and nitro) exhibit the highest inhibitory activity (Hsp90 cleavage and cell death). The biological activity of the series mainly relies on their redox capacity and their lipophilicity, which both modulate the ability of these compounds to induce a cytotoxic effect in K562 cells. As observed with other redox cycling quinones, the protein cleavage is blocked in the presence of N-terminal Hsp90 inhibitors suggesting that the availability or occupancy of nucleotide binding site in the N-terminal pocket of Hsp90 plays a critical role. In addition the survival of cancer cells and their metabolic and redox homeostasis were strongly impaired by the presence of ascorbate. Since these effects were similar to that obtained by ascorbate/menadione and they were blocked by the antioxidant N-acetylcyteine (NAC), it appears that oxidative stress is a major component of this cytotoxicity.

Combined inhibition of Wee1 and Hsp90 activates intrinsic apoptosis in cancer cells

Heat shock protein 90 (Hsp90) is an essential, evolutionarily conserved molecular chaperone. Cancer cells rely on Hsp90 to chaperone mutated and/or activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is frequently cytostatic in nature, and efforts to enhance the antitumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In agreement with previous data obtained using Wee1 siRNA, we show that dual pharmacologic inhibition of Wee1 tyrosine kinase and Hsp90 causes cancer cells to undergo apoptosis in vitro and in vivo. Gene expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional downregulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins, as well as activated Akt, was completely abrogated. These data support the hypothesis that Wee1 inhibition sensitizes cancer cells to Hsp90 inhibitors; they establish combined Wee1/Hsp90 inhibition as a novel therapeutic strategy; and they provide a mechanistic rationale for enhancing the pro-apoptotic activity of Hsp90 inhibitors.

Abstract 2773: HSP90 inhibitors target KRAS mutant human tumors through degradation of STK33

Abstract Mutations in the KRAS proto-oncogene occur in ∼30% of human cancers and are particularly prevalent in adenocarcinomas of the pancreas, lung, and colon. Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS have not met with success. We have recently shown that cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, supporting STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33, which remain to be elucidated, are not yet available. Using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex and BAG2, a member of the BAG family of molecular chaperone regulators, bind to and stabilize STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon cancer-initiating cells harboring mutant KRAS. Similar to HSP90 inhibition, short hairpin RNA-mediated suppression of BAG2 depleted STK33, whereas BAG2 overexpression rescued STK33 protein levels in the presence of HSP90 inhibitors, resulting in diminished apoptosis in KRAS mutant cancer cells. These findings (1) provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, (2) indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, (3) identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential, and (4) point to BAG2 as a candidate cancer drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2773. doi:1538-7445.AM2012-2773

Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential.

Heat shock protein 90 is a promising target for effective growth inhibition of gastrointestinal neuroendocrine tumors

Treatment of gastroenteropancreatic neuroendocrine tumors (GEP-NET) is still unsatisfactory and innovative therapeutic approaches are urgently needed. Heat shock protein 90 (Hsp90) is overexpressed in a wide range of tumor types and is an emerging target for the treatment of cancer. However, the potential activity of Hsp90 inhibitors in GEP-NET has not yet been investigated. We studied the antineoplastic activity of the Hsp90 inhibitor IPI-504 on GEP‑NET cells, and characterized its mechanism of action. In human insulinoma (CM) and pancreatic carcinoid (BON) cells IPI-504 induced a dose-dependent growth inhibition by almost 70%. The antiproliferative effect of IPI-504 correlated with a reduction in protein levels of the IGF-1 receptor. Additionally, several proteins of the PI3K/AKT/mTOR pathway, downstream of IGF-1 receptor activation in GEP-NETs, were downregulated as a consequence of Hsp90 inhibition. Combination treatment of IPI-504 with mTOR- or AKT-inhibitors led to additive antiproliferative effects. In addition, effects of IGF-1 receptor tyrosine kinase inhibition were strongly enhanced by IPI-504. Cancer gene expression profiling and FACS analysis revealed that IPI-504 antiproliferative effects were due to both induction of cell cycle arrest and apoptosis. A modified chick chorioallantoic membrane (CAM) assay confirmed the antineoplastic activity of IPI-504 in GEP-NETs in vivo. In conclusion, this study showed that Hsp90 inhibition may be an attractive target for innovative GEP-NET treatment alone or in combination with either IGF-1R or mTOR inhibitors.

Heat Shock Proteins: A Potential Anticancer Target

Heat shock proteins (Hsp) are highly conserved proteins and their expression is dependent on the level of various cellular stresses. Hsp work as a molecular chaperon for several cellular proteins and have cytoprotective roles. Their function is essential for normal cell viability and growth. Hsp90 interacts with proteins mediating cell signaling involved in essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. The naturally occurring Hsp90 inhibitor geldanamycin (GA) was the first to demonstrate anticancer activity but its significant toxicity profile in pre-clinical models precluded its clinical development. Subsequent, several Hsp90 inhibitors have been developed and underwent clinical development with favorable safety profiles. Several initial clinical studies have shown promising anticancer activity of Hsp90 inhibitors mainly in breast cancer, non small cell lung carcinoma (NSCLC), gastrointestinal stromal tumors (GIST) and various hematological malignancies. The universal involvement of Hsp90 in multiple oncogenic processes makes Hsp90 inhibitors ideal compounds to be explored as a single agent or in combination with other anticancer therapies.

Heat Shock Protein 90 Inhibitor Is Synergistic with JAK2 Inhibitor and Overcomes Resistance to JAK2-TKI in Human Myeloproliferative Neoplasm Cells

We determined the activity of hsp90 inhibitor, and/or Janus-activated kinase 2 (JAK2) tyrosine kinase inhibitor (TKI), against JAK2-V617F-expressing cultured mouse (Ba/F3-JAK2-V617F) and human (HEL92.1.7 and UKE-1) or primary human CD34(+) myeloproliferative neoplasm (MPN) cells. Following exposure to the hsp90 inhibitor AUY922 and/or JAK2-TKI TG101209, the levels of JAK2-V617F, its downstream signaling proteins, as well as apoptosis were determined. Treatment with AUY922 induced proteasomal degradation and depletion of JAK2-V617F as well as attenuated the signaling proteins downstream of JAK2-V617F, that is, phospho (p)-STAT5, p-AKT, and p-ERK1/2. AUY922 treatment also induced apoptosis of HEL92.1.7, UKE-1, and Ba/F3-hJAK2-V617F cells. Combined treatment with AUY922 and TG101209 caused greater depletion of the signaling proteins than either agent alone and synergistically induced apoptosis of HEL92.1.7 and UKE-1 cells. Cotreatment with AUY922 and TG101209 also induced significantly more apoptosis of human CD34(+) MPN than normal hematopoietic progenitor cells. As compared with the sensitive controls, JAK2-TKI-resistant HEL/TGR and UKE-1/TGR cells exhibited significantly higher IC(50) values for JAK2-TKI (P < 0.001), which was associated with higher expression of p-JAK2, p-STAT5, p-AKT, and Bcl-xL, but reduced levels of BIM. Unlike the sensitive controls, HEL/TGR and UKE/TGR cells were collaterally sensitive to the hsp90 inhibitors AUY922 and 17-AAG, accompanied by marked reduction in p-JAK2, p-STAT5, p-AKT, and Bcl-xL, with concomitant induction of BIM. Findings presented here show that cotreatment with hsp90 inhibitor and JAK2-TKI exerts synergistic activity against cultured and primary MPN cells. In addition, treatment with hsp90 inhibitor may overcome resistance to JAK2-TKI in human MPN cells.

Synthesis and biological evaluation of arylated novobiocin analogs as Hsp90 inhibitors

Novobiocin analogs lacking labile glycosidic ether have been designed, synthesized and evaluated for Hsp90 inhibitory activity. Replacement of the synthetically complex noviose sugar with simple aromatic side chains produced analogs that maintain moderate cytotoxic activity against MCF7 and SkBR3 breast cancer cell-lines. Rationale for the preparation of des-noviose novobiocin analogs in addition to their synthesis and biological evaluation are presented herein.

HSP90 Inhibition Is Effective in Breast Cancer: A Phase II Trial of Tanespimycin (17-AAG) Plus Trastuzumab in Patients with HER2-Positive Metastatic Breast Cancer Progressing on Trastuzumab

HSP90 is a chaperone protein required for the stability of a variety of client proteins. 17-Demethoxygeldanamycin (17-AAG) is a natural product that binds to HSP90 and inhibits its activity, thereby inducing the degradation of these clients. In preclinical studies, HER2 is one of the most sensitive known client proteins of 17-AAG. On the basis of these data and activity in a phase I study, we conducted a phase II study of 17-AAG (tanespimycin) with trastuzumab in advanced trastuzumab-refractory HER2-positive breast cancer. We enrolled patients with metastatic HER2(+) breast cancer whose disease had previously progressed on trastuzumab. All patients received weekly treatment with tanespimycin at 450 mg/m(2) intravenously and trastuzumab at a conventional dose. Therapy was continued until disease progression. The primary endpoint was response rate by Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Thirty-one patients were enrolled with a median age of 53 years and a median Karnofsky performance status (KPS) of 90%. The most common toxicities, largely grade 1, were diarrhea, fatigue, nausea, and headache. The overall response rate was 22%, the clinical benefit rate [complete response + partial response + stable disease] was 59%, the median progression-free survival was 6 months (95% CI: 4-9), and the median overall survival was 17 months (95% CI: 16-28). This is the first phase II study to definitively show RECIST-defined responses for 17-AAG in solid tumors. Tanespimycin plus trastuzumab has significant anticancer activity in patients with HER2-positive, metastatic breast cancer previously progressing on trastuzumab. Further research exploring this therapeutic interaction and the activity of HSP90 inhibitors is clearly warranted.

Discovery of a macrocyclic o-aminobenzamide Hsp90 inhibitor with heterocyclic tether that shows extended biomarker activity and in vivo efficacy in a mouse xenograft model

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research, heterocycle-containing tethers were explored with the intent to further improve potency and minimize hERG liabilities. This effort culminated in the discovery of compound 10, which efficiently suppressed proliferation of HCT116 and U87 cells. This compound showed prolonged Hsp90-inhibitory activity at least 24h post-administration consistent with elevated and prolonged exposure in the tumor. When studied in a xenograft model, the compound demonstrated significant suppression of tumor growth.

Abstract 623: CLU inhibition using OGX-011 is a new adjuvant therapeutic strategy for HSP90 inhibition in prostate cancer

Abstract Introduction and Objective: Prostate cancer (PCa) responds initially to anti-androgen therapies, however, progression to castration resistant disease (CRPC) frequently occurs. Several small molecule inhibitors of HSP90 show promise in CRPC and other cancers. However, most HSP90 inhibitors (17-AAG or PF-04928473 and its prodrug PF-04929113) trigger the elevation of HSPs (HSP90, 70, 27 and clusterin), which lead to tumor cell survival and treatment resistance. We hypothesized that targeting clusterin (CLU) using siRNA or the antisense drug, OGX-011, may enhance HSP90 inhibitors-induced cell death in PCa. Methods: Inducible and constitutive CLU and other HSP mRNA and protein levels were measured by real-time RT-PCR and immunoblot assays. The combination of OGX-011 with PF-04928473 or 17-AAG was evaluated in vitro on LNCaP and PC3 cells growth and apoptosis. The HSP90 inhibitor PF-04929113 was evaluated in combination with OGX-011 in vivo in athymic mice bearing castration resistant LNCaP xenografts, while the combination of OGX-011 with 17-AAG was evaluated in PC3 xenografts. Results: In prostate tumor cell lines, PF-04928473 and 17-AAG induced expression of HSPs in a dose and time dependent manner, and especially CLU at RNA and protein level, by increasing HSF-1 nuclear translocation and transcription activity. In vitro, OGX-011 synergistically enhanced the activity of HSP90 inhibitors on cell growth and apoptosis with increased sub-G1 fraction and PARP cleavage. These biologic events were accompanied by decreased expression of HSPs, Akt, AR and PSA, and HSF-1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 15mg/kg), potentiated the effect of orally administered HSP90 inhibitors (PF-04929113: 25mg/kg; 17-AAG: 50mg/kg), significantly inhibiting tumor growth by 80% and prolonging survival in PC3 and castrate resistant LNCaP xenograft model compared to the HSP90 inhibitors alone. Conclusions: These results indicate that HSP90 inhibitor-mediated induction of CLU expression can be attenuated by OGX-011, with synergistic effects on delaying progression of CRPC. This work was supported by the Canadian Institutes of Health Research fellowship and Association pour la Recherche sur le Cancer, France. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 623. doi:10.1158/1538-7445.AM2011-623

Abstract 3265A: GIST-associated c-Kit mutants exhibit differential affinities to hsp90 and its co-chaperone Cdc37

Abstract Background: Hsp90 inhibitors have been introduced to clinical treatment of imatinib-resistant GIST, but their efficacy is dependant on KIT mutations. We analysed the role of the chaperone Hsp90 as stabilizing factor for wild-type and oncogenic, misfolded c-Kit. Methods: the intracellular part of wild-type and mutated c-Kit (aa544-977) fused with an N-terminal TAP-tag was expressed in HEK293T cells. The clinically most abundant primary and imatinib-resistant secondary c-Kit mutations were selected: V560D; V559D; del557,558; L576P; K642E; V560D+D820A; del557,558+Y823D; D820A. A shortened TAP assay followed by LC-MS/MS was applied for identification of interaction partners. For verification a GST pulldown assay was used. Relative quantifications of mutation-specific interaction partners were performed by quantitative immunoblotting using an antibody raised against the C-terminus of human c-Kit. In addition, two GIST cell lines containing an imatinib-resistant and an imatinib-sensitive c-Kit mutation were investigated by quantitative immunoblotting. The activityof hsp90 inhibitor 17-AAG was studied in whole cell lysates of HEK293T cells expressing selected mutated c-Kit constructs. Results: Using the TAP-c-Kit constructs for a shortened TAP assay, Hsp90 and its cochaperone Cdc37 were identified as specific interaction partners for the GIST-associated c-Kit mutants. This interaction was confirmed using GST-Cdc37 pulldown analysis. Differential analysis of c Kit/Hsp90 interaction revealed mutation-specific affinities (up to a relative factor of about 8), with D820A showing the lowest, close-to wild-type affinity, and L576P showing the highest affinity. Among the two GIST cell lines studied, the imatinib-resistant mutant (c-Kit K642E) exhibited a 2.5 fold higher affinity to Hsp90 compared to the imatinib-sensitive mutant (c-Kit V560D+D820A). In both cell lines, the Cdc37 and Hsp90 affinities are observed in parallel. A closely related situation was observed for the interactions of the corresponding TAP-c-Kit constructs. In addition, the influence of the When analysing hsp90 inhibitor 17AAG (17-(allylamino)-17-demethoxygeldanamycin)) incells expressing selected mutated c-Kit constructs, western blot analyses showed significant degradation of total c-Kit relative to untreated controls in all mutants studied. Amongst these, mutant D820A was found to be less sensitive to 17AAG. Wild-type c-Kit showed no sensitivity against 17AAG. Conclusion: this study demonstrates mutation-specific affinities between Hsp90 and Cdc37 with c-Kit mutants. It also points to a conformity between this phenomenon and the 17AAG effect. These results allow a better understanding of the potential of hsp90 inhibition in GIST therapy and better selection of candidates for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3265A. doi:10.1158/1538-7445.AM2011-3265A

Macrocyclic lactams as potent Hsp90 inhibitors with excellent tumor exposure and extended biomarker activity

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research in this area, macrocyclic amides and lactams were explored to reduce the risk of hERG liabilities. This effort culminated in the discovery of compound 38, which showed a favorable in vitro profile, and efficiently suppressed proliferation of several relevant cell lines. This compound showed prolonged Hsp90-inhibitory activity at least 24 h post-administration, consistent with elevated and prolonged exposure in the tumor.

Inhibitors of heat shock protein 90 activity: A novel class of tumor radiosensitizers

The 90 kDa heat shock protein (HSP90) is one of major chaperones of eukaryotes which catalyzes maturation and activation of its client proteins. Among the identified client proteins there are oncogene products, hormone or growth factor receptors and key components of signaling pathways responsible for the malignant growth of tumors or their resistance to chemotherapy and radiotherapy. In the case of inhibition of the HSP90 chaperone function, such proteins are inactivated and degraded soon that leads to simultaneous blocking several pathways essential for proliferation and survival of malignant cells; therefore, pharmacological inhibitors of the HSP90 chaperone activity could be used in anticancer therapy. At present, several HSP90 inhibitors are in preclinical testing or I-III Phase clinical trials as mono-agents or in combinations with other anticancer drugs or radiation. In the present review, all the data are summarized which characterize HSP90 inhibitors as effective radiosensitizers of tumor cells. Molecular mechanisms and selectivity of the radiosensitizing action of HSP90 inhibitors are here discussed as well as a possibility of their application to improve the outcome of radiotherapy.